Infectious diseases are the major cause of morbidity and mortality, accounting for a third of the deaths which occur in the world each year. In addition, infectious agents are directly responsible for at least 15% of new cancers, and they also seem to be involved in the pathophysiology of several chronic diseases (e.g. inflammatory, vascular and degenerative diseases). Traditional infectious diseases are also highly expensive in terms of health-associated costs of infected patients and loss in productivity at work.
The main strategies used to prevent infectious diseases are therapy and prophylaxis. Vaccination has become the most cost-effective measure to prevent infections. However, there are still many diseases for which vaccines are not yet available or the available vaccines are not completely satisfactory due to low efficacy, high reactogenicity, poor stability and/or high costs. Thus, there is still an urgent need for both new and improved vaccines.
Despite the fact that vaccines have traditionally been used for the prophylaxis of infectious diseases, recent findings suggest that they are also a powerful tool for the immunotherapy of transmissible diseases (e.g. viral hepatitis, Helicobacter pylori infections, herpes virus infections, etc.). In addition, vaccines can be used for the immune-therapy or immune-prophylaxis of autoimmune diseases, inflammatory diseases, tumours, allergies and for the control of fertility in human and/or animal populations. In particular, the last application seems to require the elicitation of efficient mucosal responses at the level of the reproductive tract.
Most infectious diseases are either restricted to the mucosal membranes or the etiologic agents need to transit the mucosa during the early steps of the infection. Therefore, it is desirable to obtain not only a systemic, but also a local mucosal immune response as a result of vaccination, thereby blocking both infection (i.e. colonization) and disease development. This may result in a more efficient protection against infection, facilitating also the eradication of diseases for which humans are the only reservoirs (i.e. blocking transmission to susceptible hosts). Parenterally-administered vaccines mainly stimulate systemic responses, whereas vaccines administered by a mucosal route mimic the immune response elicited by natural infections and can lead to efficient mucosal and systemic responses. Due to the apparent compartimentalization of the systemic and mucosal immune system, parenterally administered vaccines are less effective in protecting against mucosal pathogens (McGhee, J. R., Mestecky, J., Dertzbaugh, M. T., Eldridge, J. H., Hirasawa, M. and Kiyono, H. (1992) The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 10, 75-88). Thus, administration of immunogens through the mucosal route is required to achieve full protection. However, most of the available vaccines are administered through the parenteral route, thereby, eliciting a systemic immunity in the individual.
The administration of vaccines via the mucosal route offers several advantages over parenteral vaccination. These advantages include an ease of administration, the possibility of self-administration (e.g. by intranasal, rectal or oral application), the elimination of the chance of unwanted cross-infection due to the use of infected needles or non-sterile working, lower rates of side effects, higher acceptance by the public, better compliance of vaccination protocols (i.e. increment in the overall efficacy), simpler administration logistics and lower delivery costs, being particularly suitable for mass immunization programmes. However, the compartmentalisation at the level of the mucosal immune system has to be taken into consideration. In fact, immune responses which can be observed following intra-nasal vaccination may not necessarily occur after oral or intra-rectal immunisation. For example, oral vaccination may not stimulate efficient responses in the genitourinary and/or respiratory tracts.
Unfortunately, the delivery of antigens by the mucosal route is associated with a major problem, namely that antigens delivered by this route are generally poorly immunogenic. This is the result of different mechanisms, such as (i) accelerated antigen elimination by the non specific host clearance mechanisms (e.g. ciliar activity, peristaltism), (ii) antigen degradation by local enzymes, (iii) antigen alteration and/or structural modification as a result of extreme pH (e.g. acidic in the stomach, alkaline in the intestine), (iv) poor antigen penetration through the mucosa, (v) limited access of vaccine antigens to antigen presenting cells, and (vi) local peripheral tolerance.
To overcome these problems, different strategies have been used, such as antigen entrapment or association with physical or biological particles (e.g. microparticles, nanoparticles, bacterial ghosts), the use of virosomes or viral-like-particles, the use of liposomes or ISCOMS, the use of transgenic plants, antigen production by attenuated viral or bacterial carriers acting either as conventional vectors or as carriers for nucleic acid vaccines and/or their administration with mucosal adjuvants. However, despite the heavy body of experimental evidence generated in pre-clinical studies during the last years, almost no candidates have been transferred to the vaccine development pipeline.
The use of optimal adjuvants plays a crucial role in vaccination. Antigens administered without adjuvant only rarely mediate an adequate immune response. In addition, not only the strength but also the quality of the elicited immune response matters. Stimulation of an incorrect immunization pattern may lead to immunopathological reactions and exacerbation of the symptoms of infection. In this context, the adjuvant can help to assist the desired immune response. In other words, an adjuvant can modulate the immune response or redirect the immune response to balance the immune response in the desired direction.
Substances referred to as “adjuvants” are those which are added and/or co-formulated in an immunization to the actual antigen (i.e. the substance which provokes the desired immune response) in order to enhance the humoral and/or cell-mediated immune response (“Lexikon der Biochemie und Molekularbiologie”, 1. Band, Spektrum, Akademischer Verlag 1995). That is, adjuvants are compounds having immunopotentiating properties, in particular, when co-administered with antigens. The use of many adjuvants is based solely on experience, and the effect can neither be accurately explained nor predicted. The following groups of adjuvants are traditionally used in particular: aluminum hydroxide, emulsions of mineral oils, saponins, detergents, silicon compounds, thiourea, endotoxins of gram-negative bacteria, exotoxins of gram-positive bacteria, killed or attenuated living bacteria or parts thereof.
An overview over the presently known mucosal adjuvants and delivery systems, e.g. the above mentioned particles, ICOMS, liposomes and viral-like particles, for protein, DNA- and RNA-based vaccines is given in Vajdy et al., Immunol. Cell Biol., 2004, 82, 617-627. Therein the currently available approaches in immunopentiation of mucosal vaccines are discussed.
That is, various mucosal adjuvants have been described which should serve as an alternative for the adjuvants useful for systemic administration, e.g. see Vajdy et al., supra. These mucosal adjuvants include heat labile enterotoxin and detoxified mutants thereof. In particular, genetically detoxified mutants of heat labile enterotoxin of E. coli have been developed as useful mucosal adjuvants. Moreover, cholera toxin of vibrio cholera is known as an adjuvant useful for mucosal vaccination. Further, the application of unmethylated CpG dinucleotides has been described. It was shown that CpG can bias the immune response towards a Th1 response and can modulate pre-existing immune responses. Saponins are also described as immunomodulatory substances, predominantly via the induction of specific cytokines which then modulate and/or activate the immune response.
In addition, as adjuvants which may be useful in mucosal vaccination the following have been described:
The MALP-2 molecule and Bisaxcyloxypropylcysteine-conjugates thereof, e.g. a Bispalmitoyloxypropylcysteine-PEG molecule is known to represent potent stimulants for macrophages. The usefulness of MALP-2 as an adjuvant was shown previously, see e.g. WO2004/009125 and WO2003/084568. In particular, it was demonstrated that MALP-2 can act as an effective mucosal adjuvant enhancing the mucosal immune response, e.g. fostering an enhanced expression of antigen-specific IgA antibodies.
Furthermore, it was shown that MALP-2 can activate dendritic cells and B-cells, both play an important rule in the induction of a specific humoral immune response. In addition preliminary studies demonstrate that a combination for biologically active HIV-1 tat protein and synthetic MALP-2 may be a promising vaccine with the MALP-2 component as an effective mucosal adjuvant.
Unfortunately, most of the compounds described above being useful as mucosal adjuvants are not utilisable due to their intrinsic toxicity, e.g. retrograde homing to neuronal tissues of bacterial toxoids and/or toxins at/in the derivatives after nasal vaccination.
Thus, none of these previously described mucosal adjuvants have been approved yet, but, today, only two systemic adjuvants received approval to be administered to humans and, hence, are used for the preparation of human vaccines. These adjuvants are Alum and MF59. However, both are not effective as mucosal adjuvants.
There has been an intensive search in recent years for novel adjuvants, including those for the mucosal administration route. Only a few substances have been found to be able to enhance mucosal responses. Among these, some act as carriers to which the antigens must be bound or fused thereto. Far fewer universally employable “true” adjuvants which are admixed to the antigens have been found, as outlined above.
Typically cell membranes are inter alia composed of lipids, like phospholipids. Roughly, phospholipids can be divided in phosphoglycerides and sphingolipids. The backbone of a sphingolipid is sphingosine. In all sphingolipids, the amino group of sphingosine is acylated to form a ceramide (N-acylsphingosine). The terminal hydroxyl group is also substituted. Thus, depending on the substituent a sphingomyelin, a cerebroside or a ganglioside is formed. In cerebrosides a glucose or galactose is linked to the terminal hydroxyl group of ceramide while in gangliosides an oligosaccharide is linked to the ceramide by a glucose residue.
Alpha-galactosylceramide (alpha-GalCer) as an example of alpha-hexosylceramide (alpha-HexCer) was originally isolated form the marine sponge Agelas mauritianus (Morita M., et al, J. Med. Chem., 1995, 38(12), 2176-2187). In particular, the compound Agelasphin-9b, (2S,3S,4R)-1-O-(alpha-D-galactopyranosyl)-16-methyl-2-[N—((R)-2-hydroxytetracosanoyl)-amino]-1,3,4-heptadecanetriol, is described as a potent antitumor agent. It is known that alpha-GalCer can enhance the protective immunity and displays immunomodulatory functions. Furthermore, it is described in the art that in vivo administration of alpha-GalCer leads to a potent activation of NKT-cells in mice, thus, initiating cytokine secretion, up-regulation of surface receptors and further activation of various cells of the innate and adaptive immune response. Additionally, it is speculated that alpha-GalCer has a therapeutic activity against tumors, infections and autoimmune diseases.
Alpha-galactosylceramide is able to bind to the CD1d molecule present in a subset of lymphocytes. Upon binding to CD1d, alpha-GalCer was demonstrated to activate murine and human NKT cells by recognition via antigen receptors expressed on said cells. Furthermore, it was demonstrated that nearly complete truncation of the alpha-GalCer acyl chain from 24 to 2 carbons does not significantly affect the mouse NKT cell response. Thus, the glycosyl moiety seems to be important for CD1d/GalCer and antigen receptor recognition and modification of said moiety is likely to influence binding and activation activity of alpha-GalCer.
Recently it has been described that glycosylceramides are useful as adjuvants for vaccines against infections and cancer, WO03/009812. In this document subcutaneous administration of alpha-galactosylceramides has been used to show enhancement and prolongation of malaria-specific T cell responses. Further, in WO2004/028475 the use of glycosylceramide analogues is shown. It is described that these analogues are able to immunomodulate the immune response, i.e. may activate or stimulate the immune response or, on the other hand, can have immunoinhibitory activity.
However, the use of alpha-GalCer or other glycosylceramides is limited in view of its stability and its tolerance towards the individual. Furthermore, the solubility of alpha-GalCer in aqueous solvents is poor and degradation due to enzymatic cleavage rapidly occurs. In addition, excretion of these compounds is rapid and, thus, higher dosage of said compounds is necessary.
PEGylation (i.e. the attachment of polyethylene glycol to proteins and drugs) is an upcoming methodology for drug development and it has the potential to revolutionise medicine by drastically improving the pharmacokinetic and pharmacodynamic properties of the administered drug [Parveen S, Sahoo S K. Clin Pharmacokinet 2006; 45(10):965-88.]. Since several years polyethylenglycole [is already used as a non-absorbable marker [Isenberg J I, Hogan D L, Koss M A, Selling J A. H, Gastroenterology 1986; 91(2):370-8], for the control of passive mucosal permeability (evaluated with a low-molecular-weight substance PEG 200) [Ventura U, Ceriani T, Moggio R., Scand J Gastroenterol Suppl 1984; 92:55-8] or as molecular weight marker (i.e., PEG 4000, FITC-dextran 10.000). It was demostrated, that PEG showed only low intranasal irritation in humans [EP 0532546] and also low toxicity was found in rabbits or in sheeps after 1 repeated nasal application (three times a day) of pure PEG. The usage of pegylated immuno-nanoparticles synthesized with bifunctional PEG derivatives showed that these component can link the nanoparticle with the targeting MAb [Olivier J C, Huertas R, Lee H J, Calon F, Pardridge W M., Pharm Res 2002; 19(8):1137-43].
However, the use of pegylated compounds, such as the current standard therapy for HCV, pegylated interferon alpha in combination with ribavirin, has its limitations. Limited efficacy in patients with hepatitis C virus genotype 1 and the side effect profile will necessitate the development of new therapeutic approaches [Manns M P, Wedemeyer H, Cornberg M, Gut, 2006; 55(9), 1350-9]. Furthermore, the conjugation of an immunomodulator with PEG does not matter, that the pegylated compound still is able to act as an adjuvant. Studies with pegylated Malp-2 derivatives showed a decrease in cellular proliferation and also in the secretion of antigen-specific IgG titer in comparison to Malp-2. Until now, it has not been demonstrated, that a pegylated derivative of a chemical active compound was able to stimulate and activate an antigen-specific immune response via intranasal administration route. The usage of the conjugates according to the present invention, e.g. the new αGalCerMPEG compound as systemic, but also as mucosal adjuvant showed that said conjugates are able to enhance antigen specific immune responses without adverse side effects.
Hence, there is still a need in the prior art to provide new compounds useful as adjuvants, particularly as mucosal adjuvants and/or as vaccines overcoming the drawbacks mentioned above, in particular, having good stability and tolerance in the individual while being soluble in aqueous solvents, being protected against degradation in the individual and with good shelf life. In particular, there is a need for mucosal adjuvants which can elicit a strong immune response which represent a balanced or adjusted immune response involving both humoral and cellular components, thus, allowing effective prophylaxis or treatment of various diseases and conditions, specifically of infectious diseases or cancer.
Thus, the object of the present invention is the provision of mucosal adjuvants which can elicit and/or enhance and/or modulate (pre-existing) immune response in an individual or subject. In particular, the invention was based on the object of developing a range of novel, highly active adjuvants, particularly mucosal adjuvants which are non-toxic for humans and which can be employed with a wide variety of active ingredients to be assisted in conventional or novel vaccines such as, in particular, prophylactic or therapeutic vaccines, including cancer and DNA vaccines.